Ceramide 2 (N-acetyl sphingosine) is associated with reduction in Bcl-2 protein levels by Western blotting and with apoptosis in cultured human keratinocytes

Background Ceramides produced by sphingomyelin hydrolysis activate a cycle that is followed by three different major cellular responses: downregulatio n of cell proliferation, induction of cell differentiation and apoptosis. I n the skin, the generation of intracellular ceramide may also provide a lin k between an extracellular signal and the induction of the apoptosis progra mme for the elimination of damaged cells. Objectives We investigated the effect of ceramides capable of entering cell s on cultured keratinocytes. Methods Human keratinocytes from neonatal skin were cultured in serum-free medium with or without increasing concentrations of ceramide 2 (CER-2; N-ac etyl sphingosine) (5, 10, 20 and 40 mu mol L-1). Proliferative effects were studied either by cell counts or by H-3-thymidine incorporation and flow c ytometric analysis. Apoptosis was studied by TUNEL staining and Western blo t analysis of Bcl-2 protein. Results Cell counts and DNA synthesis were reduced in a dose-dependent mann er following CER-2 treatment. TUNEL staining showed CER-2-induced apoptosis at 48, 72 and 96 h. Western blot analysis showed that CER-2 induces downre gulation of Bcl-2, at 24-96 h. Conclusions These results demonstrate that CER-2 inhibits cell proliferatio n and induces apoptosis, possibly via a Bcl-2-dependent mechanism.